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Pests of animals 2. TITLE AND SUBTITLE A sequential study of the pathogenesis of disease caused by
Trypanosoma vivax in experimentally infected calves,utilizing clinicalpathological, histopathologicaland immunofluorescent techniques 3. AUTHOR(S)
Daley,C.A.
6. ARC NUMBER4. DOCUMENT DATE S. NUMBER OF PAGES
1971 . ARC 7. REFERENCE ORGANIZATION NAME AND ADDRESS
Tex.A&M
8. SUPPLEMENTARY NOTES (Sponeorlng Organizatlon,Publahere,Availability)
(Thesis M.S.--Tex.A&M)
9. ABSTRACT
10. CONTROL NUMBER 11. PRICE, OFDOCUMENT
PN-RAA- 116
12. DESCRIPTORS 13. PROJECT NUMBER
Antibodies ,bacterial Trypanosoma I
Fluorescence Trypanosomiasisbovine 14. CONTRACT NUMBER
Histological technics CSD-1947 Res. Pathology 15. TYPE OF DOCUMENT
AID 590-1 (4-74)
A SEQUENTIAL STUDY OF THE PATHOGENESIS OF DISEASE
CAUSED BY TRYPANOSOMA VIVAX IN EXPERIMENTALLY
INFECTED CALVES, UTILIZING CLINICAL, PATHOLOGICAL,
HISTOPATHOLOGICAL AND INKUNOFLUORESCENT TECHNIQUES
A Thesis
by
CHARLES ARTHUR DALEY
Submitted to the Graduate College of the Texas A&M University in
partial fulfillment of the requirement for the degree of
MASTER OF SCIENCE
Nay 1971
Major Subject: Veterinary Pathology
A SEQUENTIAL STUDY OF THE PATHOGENESIS OF DISEASE
CAUSED BY TRYPANOSOMA VIVAX IN EXPERIMENTALLY
INFECTED CALVES, UTILIZING CLINICAL, PATHOLOGICAL,
HISTOPATHOWGICAL AND INMUNOFLUORESCENT TECHNIQUES
A Thesis
by
CHARLES ARTHUR DALEY
Approved as to style and content by:
(Chairman ol1Coumttee)
(edo eatinent) (Mimber)
(Member) (Member)
((Member)
May 1971
ABSTRACT
A Sequential Study of the Pathogenesis of Disease Caused by
Trypanosoma vivax in Experimentally Infected Calves, Utilizing
Clinical, Pathological, Histopathological and Immunofluorescent
Techniques. (May 1971)
Charles Arthur Daley, B. S., H. S., Montana State University,
D. V. H., Washington State University
Directed by: Dr. Charles H. Bridges
Trynanosoma vivax obtained from a clinically sick cow near
Neiva, Colombia, was passed in a sheep and a calf and inoculated
into the jugular vein of 14 Holstein-Friesian calves. Fever oc
curred by 24 hours, and recurring parasitemia commenced after 72
hours. It was estimated that practically all of the 14 calves
would have died spontaneously within 3 months if none had been
euthanitized. Associated with the first and subsequent parasi
temias were decreases in hemoglobin, PCV, M:E ratio, serum
albumin, A:G ratio and neutropenia.
All calves exhibited gradual weight loss by 2 weeks and
later submandibular edema usually became evident. Consistent post
mortem lesions seen after 4 weeks were conspicuously hypertrophied,
edematous lymph nodes, hypertrophied hemal lymph nodes, emacia
tion, rounded right heart, palpably firm liver, atrophied thymus
and hypertrophied femoral bone marrow.
Associated with T. vivax of the infecting inoculumand,
iv
succeeding parasitemias were generalized endothelial hypertrophy
and mononuclear cell infiltration along blood and lymph vessels
with proteinuria and bone marrow hyperplasia. Generalized lympha
tic-and RE'hyperplasia occurred, but was not proven to be due to
At 3 weeks there were aggregations ofthe trypanosomiasis.
macrophages containing engulfed material distributed along
capillaries in pulmonary interalveolar tissue, and this lesion
in combination with the anemia and apparent cardiac insufficiency
were thought important in the development of anoxia, and pro-
Periacinarbably contributed to the single fatality observed.
congestion and fatty metamorphosis of midzonal hepatocytes were
probably related to the failing heart and indirectly to the pul
monary lesion. Cystitis and pyelonephritis in the last 2 calves
to be killed probably was a reflection of generalized, chronic
debility.
The detection of trypanosomes in histologic sections, using
a direct fluorescent antibody technique, was impeded by a gen-
It waseralized'fluorescence of tissue's 'of infected calves.
suspected thatthe fluorescence of-tissues was related to the
presence of soluble antigens which are known to exist in T. vivax
infections.
V
ACKNOWLEDGMENTS I
The completion of this research was facilitated by the
cooperation of many people from several countries.
To the committee members and other staff members of The
College of Veterinary Medicine, Texas AM University, the
author extends appreciation, especially to the committee chair
man, Dr. Charles H. Bridges, for his thorough competence in
histopathology and guidance in the research, and to Dr. L. G.
Adams, Leader of the Texas Group in Bogota', Colombia. Dr.
Adams is to be admired for his conscientious, effective teaching
efforts in 2 languages, and for his tactful, very workable and
professional relationship with people at all levels in the
handling of numerous sensitive situations where technical
excellence is only a single, small requisite to effective
solutions satisfactory to all people concerned.
Acknowledgment is made to personnel of Centro Internacional
Agricultura Tropical (CIAT), The Rockefeller Foundation in Colom
bia and Instituto Colombiano Agropecuario (ICA) and particularly
to Dr. Jaime Estupinan, Dr. Guillermo Mateus and Dr. Gustavo
Riveros for their cooperation in the research project; to Mr. Luis
Eduardo Ramirez, Miss Nelly Giron, Miss Beatrice Roitman (all of
Colombia) and Mrs. Zelma Grady (College Station, Texas) for
technical assistance; to Mr. Luis Garcia for competent calf hus
bandry; to,Mr. Joqe Leal for his professional draftinig and crafts
vi
manship as well as instruction in Spanish; and to Dr. E. A. Wells
of the United Kingdom for his aid in the isolation and identifi
cation of Trypanosoma vivax.
The author and his wife are grateful for the financial
assistance from U.S.A.I.D. and Rockefeller Foundation which made
possible the author's opportunity to receive advanced training
in veterinary pAthology at Texas AMM University and the oppor
tunity to participate in a foreign animal disease program, as
well as to live for a year in a culture unfamiliar to themselves.
The ultimate thank you is due to Dr. Fred M. Maurer, whose
tireless efforts to improve world animal protein production by
improving domestic animal health have resulted in the establish
ment of The Institute of Tropical Veterinary Medicine at Texas
A&M University, College Station, Texas.
vii
TABLE OF CONTENTS
Chapter Page
I INTRODUCTION . . . 1
II LITERATURE REVIEW . . . . . . . 2
III MATERIALS AND HETHODS . . . . . . 8
IV RESULTS# o . o 17
V DISCUSSION . . . . . . . . 37
VI SUMMARY. * a . . 0 . . 53
REFERENCES. . . . . . . . . . . 56
VITA o 0. o 0 0 61
viii
LIST OF FIGURES
PageFigure
Morning and Afternoon Rectal Temperatures andI . . . .° , 18 . .Hematological Data ,
Calf Infected 6 Weeks Previously with Trypano2 19oma vivax,*
. 21 . . . • . •3 Hematological Data (1).
. . . • 22, , ,4 Hematological Data (2)6
Lung from Calf Infected 38 Days Previously5 * , * 27, , *
Heart from Calf Infected 38 Days Previously
with Trypanosoma vivax. ,
6' , * , • 29with Trypanosoma vivax. , , ,
Liver from Calf Infected 38 Days Previously7 o 29o @ o 9 * with Trypanosoma vivax.
8 Blood Smear from Trypanosoma vivax Infected
Calf Incubated 30 Minutes (37 C) with FITC. • • 35
conjugated Immune Serum . , . •
CHAPTER I
INTRODUCTION
Bovine trypanosomiasis is caused by a flagelated protozoan
belonging to the genus Trypanosoma (29). Kubes, in 1944, pre
sented evidence that Trypanosoma vivax had spread through certain
countries and adjacent islands in Central and South America during
this century (34). His findings were based on the morphologic
similarities of organisms found there with 1. vivax isolated dur
ing the original African studies.
Wells, et al. in 1969 found evidence that clinically normal
cattle of various ages harbored T. vivax in Colombia, South Amer
ica, although the vector was not discovered (50). The tsetse fly
which is the known vector of bovine trypanosomes in Africa has
never been found in the New World.
The importance of trypanosomiasis in South American cattle
has not been established. The blood of cattle frequently contains
T. vivax as well as Anaplasma marinale and/or Babesia j. A
need therefore existed to characterize the clinical, hematologic,
biochemical and pathological aspects of trypanosomiasis alone in
cattle, and to obtain a basic understanding of the pathogenetic
process.
The citations on the following pages follow the style of American Journalof Veterinary Research.
2
C H'APTER II
LITERATURE REVIEW
The pathogenicity of the disease caused by v. vax in
African cattle is generally regarded as of major importance (35).
Fiennes stated that T. vivax often caused either a chronic or
benign disease with a high recovery rate, and only exceptionally
caused peracute or acute disease (15). Hudson reported that
T. vivax caused fever initially, and subsequently hypoglycemia
and hemorrhages in the tongue, adrenals and kidney (30). Death
occurred in the subacute stage from severe anemia rather than
hypoglycemia. In another study, Fiennes found multiple throm
boses of the smaller blood vessels in the spleen, liver, lymph
nodes, lung and adrenal glands of cattle with Trypanosoma con
golense (14). The tunica adventitia of many of the vessels of
the organs studied became hyperplastic with loss of elasticity,
and the endothelial cells reportedly underwent "metaplasia" and
passed into the general circulation.
Foci of chronic infection by both 1. conzolense and T. vivax
were found in bovine cardiac tissue (17).. The cattle had been
exposed to infected'tsetse flies and then treated with trypano
somicidal drugs. Most of the organisms were degenerated or lysed,
but some were normal, and were always found in capillaries. The
occurrence of myocardial necrosis varied, but often was severe.
Lodged parasitic'emboli were suspected to have caused local
3
Ischemia.
Most clinical pathological data from cattle infected with
. congolense or 1. vivax included decreased hemoglobin values
and erythrocyte counts (15, 30, 34, 39), eoLinopenia (16), neutro
philia (39) or neutropenia (13, 16), lymphocytosis (13, 39),
periodic monocytosis associated with a reduction in numbers of
circulating trypanpsomes (16), elevated serum glutamic-oxalacetic
transaminase (SGOT) levels (26) and no change in bilirubin levels
(139 16). Serum albumin levels decreased in sheep with T. vivax
(6, 40), in rats with Trypanosoma brucei (31) and in rabbits with
Trypanosoma evansi (47). Gamma globulins increased (6, 31, 40).
Blood glucose levels remained normal in cattle with 1. congolense
(13), but decreased in rats with :. brucei (48).
Fiennes summarized certain theories (15) as follows: Direct
intervention by the parasites can not be substantiated, because
parasites can not be demonstrated by detailed examinations.
Intracellular developmental forms have been proven to exist only
with Trypanosoma cruzi. Histologic lesions caused by intoxica
tion induced by dead trypanosomes has not been proven. An
antigen-antibody reaction causing injury to the somatic cells
has been thought to occur.
Because of the lack of information on the pathogenesis of
disease caused by 1. vivax, data on other trypanosomal diseases
has been reviewed. Results of experimental disease caused by
I. brucei in mice indicated that infectivity and pathogenicity
4
appeared to be related (37).' Both varied according to the anti-
Gordon et al. (24) exposed rabbits togenic type involved.
Trypanosoma rhodesiense from carrier insects and observed local
multiplication of the parasites, "chancre" formation and invasion
of the blood stream.
Nigerian swine infected with Trypanosoma simian developed
fever and died acutely without anemia (32). Thrombi were present
in hepatic arterioles, pulmonary arterioles, interlobular veins
of the renal cortex, arterioles at the hilus and trabeculae of
lymph nodes, small vessels of the brain, medium sized vessels of
The thrombithe adrenals and submucosal vessels of the intestine.
contained fibrin, mononuclear cells and in some cases whole or
fragmented trypanosomes. Endothelial cells of several thrombosed
capillaries were hypertrophic.
That trypanosomes are influenced by body temperature was
suggested by a report from India in which the parasites were
found chiefly in the skin capillaries of cattle infected with
Fiennes found 3. congolense infections in the skinT evansi (1).
of cattle and mentioned the rapid removal of the parasites once
they entered the main circulation (14). He stated that serum
antibody titers and body temperatures above 38 C were probably
prime 2actors in the defense mechanism. Markinelle studied highly
pathogenic T. cruzi infections in mice and found that a high
(36 C) environmental temperature may protect mice for months (36).
Rabbits infected with Trynanosoma gambiense developed skin
5
lesions containing high concentrations of trypanosomes before
death (45). A vascular permeability-increasing factor was de
monstrated in aqueous extracts of the parasite when inoculated
intradermally following systemic introduction of Evans' blue dye.
T. brucei was studied in rabbits using ear chambers, contrast
media with X-ray films and India ink (23). The endothelium of
venules and capillaries became irregular and blood flow was im
peded. Many large macrophages which lined the vessels contained
carbon particles. Trypanosomes appeared cyclically in spaces
between vessels, and were often seen when the parasitemia was low.
Just before death the vessels became "sticky" and were lined with
leucocytes. When vascular circulation ceased, the vessels dis
integrated within a few hours. Fiennes described vascular lesions
in the bovine species that resembled the vascular lesions in
rabbits (14).
It has been established that mammalian hosts develop anti
body titers against trypanosomes, and that in some unknown way,
the antigenic character of the trypanosomes is altered (27, 40, 49).
The ability of trypanosomes in mammalian hosts to change in anti
genic character has been offered as an explanation for the fluctu
ations in parasitemias which are commonly observed. Recently it
has been reported that although chick embryos infected with .
rhodesiense or 1. brucei developed neither agglutinating
antibodies nor antibodies detectable with fluorescent antibody
techniques, fluctuations in trypanosome numbers occurred (22).
6
Antibody levels in cattle with T. brucei have been related to
increased kinin activity following each peak of parasitemia, and
to a decrease of the precursor of kinin in the blood (4). Tha
kinins are 10 times as active as hisamine,' causing increased vaso
dilatation and capillary permeability with resulting edema and
pain.
Schroeder and Ristic demonstrated a heat-stable opsonin in
calves infected with Anaplasma marginale (44). Titers appeared to
be correlated with intensity and persistence of anemia and with
the amount of erythrophagocytosis in the bone marrow. An auto
the cause of the anemia.immunization mechanism was proposed as
Zuckerman proposed 4 models to account for the various evi
dence of autoimmunization in protozoa (52): 1. The infectious
agent may share an antigen with the host cell, with antiparasitic
antibody then combining with the heterogenetic antigen in the host
cell. 2. The target host cell might be coated with parasitic
antigen, which attracts antiparasitic antibody, followed by cell
ular damage. 3. The target host cell may be directly coated
with antiparasitic antibody and be sensitized. 4. The target
host cell may be so modified by an infectious agent, or its pro
ducts, or by a drug used in treating the infectious agent, as to
become autoantigenic. Zuckerman stated that protozoan diseases,
inwhich it was suspected that autoimmunity had occurred, were
all diseases of the blood and tissues where the organisms-had
intimateicontactwith cells, and often were intracellular in
7
nature, Intracellular forms of T. vivax have not been demonstrated
as they have with . 6ruzi, in which development occurs in a series
of mesenchymal cells, preferentially in cardiac tissue.
8
,CHAPTER III
MATERIALS AND METHODS
The isolate of 1: vivax which was used in the experiment was
obtained from a clinically ill cow near Neiva, Colombia, and was
identified by its non-infectivity for laboratory mice, by its be
havior in culture and by its morphology. The isolate was passed
once in sheep to eliminate Babesia lP. and then in a calf which
was treated with Oxytetracycline HCl* intravenously at the rate of
12 mg./kg. for 12 days and with B-W Drug No. 356-C-61** intraven
ously at the rate of 5 mg./kg. for 3 days to eliminate Anaplasma
Serum from this calf remained free of complement fixmarainale.
ing antibody titers for Anaplasma and Babesia antigens.
Eleven intact male and 4 female Holstein-Friesian calves 4
All of the calves were raised on pasturesto 6 months were used.
located in the Sabana of Bogota, an area considered to be trypano
some free. The calves were examined hematologically for trypano
somiasis and serologically for anaplasmosis and babesiosis and
found to be free of these diseases. The calves were maintained
as a group in one section of a barn, and were not allowed to mix
with other cattle. Prior to their inoculation with trypanosomes,
5 cc. of blood from each of the 15 experimental calves was
*Chas. Pfizer Co., Inc., New York, N.Y.
**Burroughs-Wellcome,Co., Research Park Triangle, N.C.
9
inoculated intravenously into a susceptible splenectomized calf,
cultured on enriched blood agar (51), inoculated intraperitoneally
into 2 laboratory mice at the rato of 1.0 cc. each, and examined
in wet mounts.
One calf served as an experimental control in which the con
tinued absence of blood parasites throughout the experiment indi
cated that contamination from outside sources was not likely.
The control calf was subjected to necropsy last, and the tissues
served as negative controls for the immunofluorescence study.
Oxylated blood from the carrier calf was examined daily, and
a relative scale for quantitating the degree of parasitemia was
established. In each test, 10 microscopic fields of fresh blood
in a wet mount slide were examined at 400 magnification. If no
trypanosomes were found, the test was considered negative, and if
1 trypanosome was found in a total of 10 fields it was designated
a "1 plus", If an average of 1 trypanosome was found in each of
10 fields it was designated a "2 plus". When there was more than
1 trypanosome in each of 10 fields it was designated a "3 plus",
and if a high number of trypanosomes were present in each of 10
fields it was designated a "4 plus".
Each of the 14 experimental calves was given intravenously
5.0 cc. of citrated blood containing approximately 100,000 organ
isms per cc. A sample of infective blood was frozen in glycerol
using the technique of Cunningham, et al. (8). The sample was
stored at -79 Clinsuring a stabilate of trypanosomes (as defined
10
by Lumsden) for future work (35).
Beginning 5 days prior to theinoculation of the calves and
continuing,throughout the experiment,'rectal temperatures were
measured each morning and afternoon. Samples of blood from each
calf were collected on each of 4 days before inoculation and sub
sequently on each day of scheduled necropsy. Alijuots of the
blood were treated with sodium fluoride or sodium oxalate and
Blood glucose concentrationsother aliquots were allowed to clot.
(38), packed cell volumes, hemoglobin concentrations, sorbitol
dehydrogenase levels (19), serum glutamic oxalacetic transaminase
concentrations (41), total serum proteins (2, 12), bilirubin con
centrations (20), total and differential leucocyte counts and
Theseserum protein electrophoretic patterns (5) were determined.
data obtained from the 14 infected calves are summarized in
Figures 1, 3 and 4. The group mean and standard deviation are
shown on each day of observation.
The packed cell volume was determined by standard laboratory
methods. The hemoglobin concentration was determined by the
Hycel* cyanmethemoglobin technique. Serum bilirbin content was
assayed by the technique described by Gibson and Goodrich (20).
Total serum protein was determined by a hand held temperature com
pensated Goldberg refractometer (A-0 TS meter).** Serum protein
*Hycel Co:, Houston 2, Texas
**American Optical TS Meter, Buffalo, N.Y.
electrophoresis pn cellulose polyacetate membranes was done using
a Gelman deluxe electrophoresis chamber, Model 51210.* Aliquots
of 5.0 lambdas of serum were applied to cellulose acetate mem
branes soaked in fresh cold barbital buffer having pH 8.6 with an
ionic strength of 0.05. Electrophoresis was continued for 45
minutes at 1.5 to'l.8 milliamperes at a constant direct current
voltage of 350 volts maintained by a Gelman voltage supply,
Model 38201.* The cellulose polyacetate membrane was stained
for 5 minutes in a solution of 0.1% Ponceau S with 5% trichlor
acetic acid, differentiated in 5% acetic acid (1 minute in each
of 3 baths) and cleared for 30 seconds in a mixture of 10% glacial
acetic acid and 90% methanol. It was dried on glass slides at
60 C for 20 minutes and quantitated by scanning on a Gelman auto
matic recording and integrating scanner, Model 39372.*
Serum glutamic oxalacetic transaminase levels were measured
by a modification of the original Reitman and Frankel method (41).
Blood glucose concentrations were determined using a photometric
adaptation of the Somogyi method (38).
One calf, selected by random sampling, was euthanatized by
electrocution at each of the post inoculation times of 12 hours,
1, 2, 3, 5, 8, 12, 17, 23, 30, 38, 45 and 88 days. One calf was
necropsied on day 37 following death due to trypanosomiasis.
The control calf was the last calf to be killed.
*Gelmata Instrument Co., Ann Arbor, Mich.
12
As each calf was dissected at necropsy, triplicate sets of
tissues were collected. One set was preserved in buffered 10%
formalin and tissue sections were stained with hematoxylin and
eosin, using standard techniques. The other 2 sets were frozen
onto wooden blocks using Cryoform* and stored in separate freezers
at -20 C for immunofluorescence studies.
Emphasis was placed on the histologic study of sections of
tissues from the major organs and lymph nodes, although tissues
from each of the following anatomical sites were examined: lip,
tongue, dental pad, ear, eye, nasal mucosa, mandibular lymph node,
mesenteric lymph node, prefemoral lymph node, hemal lymph node,
lung, heart, aorta, bone marrow, adrenal, kidney, urinary bladder,
spleen, pancreas, rumen, reticulum, abomasum, duodenum, ileum,
colon, liver, gall bladder, ovary, uterus, penis, testicle,
cerebrum, cerebellum, medulla oblongata, choroid plexus, pitui
tary, spinal cord, bone, semilunar ganglion, semitendinous muscle,
diaphragm and mediastinal lymph node. All samples of a given
organ except those samples representing gross lesions were taken
from similar locations within the organ.
Bone marrow biopsies were taken from the ribs of 3 calves on
each scheduled day of necropsy. The 3 calves included the calf
to be dissected, plus 2 other calves selected at random. After
all the calves had been subjected to I biopsy, the selection
*International Equipment Co., Needham Heights, Mass.
13
process was repeated, using ribs from the opposite sides of the
calves.
On day 23 a quantity of blood was withdrawn from an infected
calf selected at random and allowed to clot in order to obtain
serum containing antibodies against T. vivax. The 112 cc. of
serum was put in dialysis tubing with an air pocket at each end
and then placed in a 1000 cc. beaker containing half saturated
(NH4)2SO4 solution. The solution was agitated overnight with a
magnetic stirrer in a refrigerator set at 4 C. The solution was
changed twice. After 24 hours the serum volume had been reduced
to 46 cc. An equal volume of physiological saline containing 1
drop of 1.0 N NaOH per liter was added. To this was added, drop
by drop, 56 cc. of saturated (NH4 )2SO4 , using a magnetic stirrer.
After 1 hour of stirring, it was centrifuged at 10,000 rpm for
15 minutes using a refrigerated Sorvall* Model RC2-B centrifuge.
The supernatant was discarded, and the precipitate was washed with
half saturated (NH4 )2S04 . The centrifugation-wash cycle was re
peated 2 more times. The precipitate was then redissolved in a
minimum quantity of 0.5 molar Na2CO3-NaHCO3 solution at pH 9.0
(Q part 0.5 molar Na2CO3 and 4 parts 0.5 molar NaHC03 ). The re
suspended material was placed in new dialysis tubing and dialyzed
against 0.05 molar NaHCO3 solution at pH 7.5 in a 1000 cc. beaker.
The solution in the beaker was changed twice overnight. The
*Ivan Sorvall, Inc., Newtown, Conn.
14
material in the dialysis tubing, now having'a volume of 51 cc.,
contained 4.9% protein. A sample of it was examined by cellulose
acetate zone electrophoresis and contained gamma globulin with
no albumin. The 51 cc. of solution was expanded to 100 cc. by
mixing with 38 cc. of cold physiological saline and then slowly
adding 11 cc. of buffer solution pH 9.0 (Q part 0.5 molar Na2CO3
and 4 parts 0.5 molar NaHC03 ). One milligram of fluorescein iso
thiocyanate (FITC)* was used for each 20 milligrams of protein in
the solution. The FITC was dissolved in 2 cc. of acetone and was
added slowly to the swirling solution which was then stirred over
night in the refrigerator. The conjugated preparation, which had
a pH of 8.9, was passed through 2 columns containing Sephadex**
G-25 (Course), and the gamma globulin containing portion was col
lected in fractions and tested for protein concentration. It was
filtered, using a Swinny filter holder containing a Milipore***
filter with a 0.45 micron pore size, and stored in 1.0 cc.
aliquots at -20 C.
Positive control slides for the fluorescent antibody study
were prepared as follows. Blood from an infected calf having a
4-plus parasitemia was collected in tubes containing the sodium
salt of ethylenediaminetetraacidic acid (EDTA). The blood was
*Nutritional Biochemicals Corp., Cleveland, Ohio
**Pharmacia FineChemicals, Inc., Piscataway, N.J.
***Milipore Filter Corp., Bedford, Mass.
15
centrifuged at approximately 500 rpm until the supernatant was
clear. The supernatant was collected in a separate tube contain
ing a very small quantity of the erythrocytes from the original
tube. This material was mixed by swirling, and 1 drop was placed
on each glass slide, spread evenly across the surface and allowed
to air dry. The slides were fixed for 10 minutes with acetone
at -20 C and then allowed to dry. They were stored in slide box
es surrounded by plastic bags containing a small quantity of
CaCd2 in a freezer at - 20 C.
Sections of uninfected tissues were cut from frozen blocks of
mediastinal lymph node and kidney taken from the control calf.
Sections for FA studies were cut at 4 microns. Those tissues
which were examined by FA techniques included those of the ear,
cerebrum, thymus, heart, lung, mediastinal lymph node, prescapu
lar lymph node, liver, spleen, kidney and bone marrow. The sec
tions were mounted on new glass slides that had been previously
cleaned in acetone and air dried. The tissues were fixed in
acetone at -20 C.
Tissues from each infected calf were stained and examined as
a group along with at least 1 positive control slide and 1 each
of the negative control slides. The slides were allowed to warm
to room temperature. The conjugate was thawed and diluted 1:50
with 0.01 molar NaH2PO4 buffered saline with pH 7.5, and care
fully added to the tissue so that its entire area was covered.
Each preparation was placed on supports in a separate covered
Petri dish containing moistened filter paper and incubated for
30 minutes at 37 C. Then the sections were removed from the in
cubator and washed for 15 minutes in each of 3 successive changes
of phosphate-buffered saline that had been chilled previously.
The sections were air driad and cover slips were applied, using
a non-fluorescing glycertn solution at pH 9.0. They were ex
amined within 1 hour, using a Leitz Ortholux binocular microscope
equipped with lOX oculars and lOX, 40X, 54X oil immersion and
IOOX oil immersion objectives and a dark field condenser. Non
fluorescing immersion oil was employed. The light source was an
Osram Mercury super pressure HBO-20OW lamp with a diffusion fil
ter, a BG 12 excitor filter and a K 470 barrier filter.
17
CHAPTER IV
RESULTS
Clinical Manifestations
The first clinical sign was an increase in the morning rectal
temperature which averaged 2.5 C above normal (Fig. 1). The fever
was at a maximum 24 hours after inoculation and had decreased by
day 3. The average afternoon ractal temperatures are shown in
Fig. 1.
Changes in the appearance of the calves were detectable
approximately 2 weeks post inoculation. The calves gradually be
came emaciated and their hair coats became dry. At approximately
4 weeks post inoculation the calves appeared more docile, their
heads drooped, and general weakness gradually became apparent.
At 5 weeks post inoculation submandibular edema could be detected
in some calves (Fig. 2). The edema receded in all animals as
death became imminent and in 2 calves the edema had regressed
completely before death. Dehydration and lacrimation were appar
ent and the abdomens became gaunt after 5 weeks on experiment.
Appetite remained near normal, but there was a general lack of
vigor in this phase. The calves moved more sedately toward their
feed and appeared to eat in a more languid manner.
Clinical Pathology
The parasitemia became evident on day 5 and reached a
101
t-om T-n
41. .0.
10
i
'35~
13125
+ 11064'% 40 j 1
nw 0
3bA
s -3 30 35 4mW"Y
Fig. 1. Morning and afternoon rectal temperatures and hemato
logical data. Group" means are presented and vertical lines
represent t standard deviation. Number of calves decreased
because of selective serial necropsy. Arrow Indicates time of
intravenous inoculal'Aon with Trynanosoma vivax.
19
Fig. 2. Calf infected 6 weeks previously with Trypanosoma vivax.
Note submandibular edema, lacrimation and generally depressed
attitude. Death was expected following the onset of these signs.
20
maximum (4-plus level) on'day 7 (Fig. 1). Thereafter, the degree
of parasitemia in a given calf varied from zero to a,maximum re
lative value of 4-plus.
The infected calves became anemic as the experiment pro
gressed. Hemoglobin levels and packed cell volumes decreased
steadily (Fig. 1). There was an overall decrease in the myeloid:
erythroid (M:E) ratio (Fig. 1). Total and direct bilirubin levels
increased slightly, and-then fluctuated thereafter (Fig. 3). The
SGOT levels increased in the last half of the experiment and the
standard deviation increased (Fig. 3). Serum sorbitol dehydro
g,3nase levels increased and then regressed (Fig. 3).
The level of total serum proteins had decreased by day 8 but
remained constant thereafter (Fig. 3). A slight decrease in the
absolute amount of serum albumin occurred. Although there was a
relative increase in the percent of globulins, there was actually
a slight decrease in the absolute amount of globulins present due
to the decrease in total serum proteins. The albumin:globulin
(A:G) ratio decreased slightly (Fig. 3). The absolute amount of
gamma globulins increased, while,the absolute amount of alpha and
beta globulins decreased (Fig. 3).
Blood glucose levels remained normal until after day 30,
when the mean values decreased to one half those of the pre
inoculation values (Fig. 4).
Total leucocyte counts decreased by day 8 and remained
fairly constant until day 30 when there was an abrupt increase,
21
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"'5 - tllli go7~~l
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3De
are nFig0 G.Hmtlgcldt.Gopman"Arsne
F 2315 1 0 17 23 3 38 0
Pho U4mifl U V 0 D 13 1-1
Fig. 3. HemAtological data. Group means are presented and vertical lines represent t standard deviation. Number of calves decreased because of selective serial necropsy. Arrow indicates time of Lntrave .ousinoculation with Trypanosoma 'vivax.
22
--- aglcose
70-
Date of 5050
- .a Neutro
4'1
10 10
0 V
8 ~1 ~ 2 ~12 ec__0___
__ monoID go 0
Fig.~~~~~~~~~~~8 4.Hmtlgcldt.Gopmasaepeetdan vetcllnsrpeet Nuro0tnaddvain
calvs dcresedf slectve twil neropy. rroecase inicte tmofitaeosicuaonwt rasoa
vivaxE
23
to a level above normal (Fig. 4). The leucopenia was influenced
by the development of neutropenia in the acute phase (Fig. 4).
This condition persisted until near the end of the experiment,
when a neutrophilia developed.
There was a slight decrease in the number of lymphocytes by
day 8 (Fig. 4). However, there was a relative lymphocytosis due
to the considerable decrease of total leucocytes. The absolute
number of lymphocytes was approximately normal at the end of the
experiment. A very slight eosinopenia and monocytopenia devel
oped gradually throughout the course of the experiment (Fig. 4).
Gross Pathology
The tissues and organs of the calves killed at 12 hours, 1,
2, 3 and 5 days were not observed to have lesions except for an
occasional small area of consolidation in the apical or cardiac
lobes of the lungs.
In the calf killed on day 8 the lymph nodes were slightly
edematous and the hemal Xymph nodes were slightly enlarged.
The abdominal cavity of the calf killed on day 12 contained
approximately 30 cc. of amber fluid. One prescapular lymph'node
contained multiple petechiae. The thymus appeared edematous and
the spleen was slightly enlarged.
The lymph nodes were enlarged and edematous in the calf
killed on day 17. The hemal lymph nodes were slightly enlarged.
The thymus was reduced in size.
24
The lymph nodes of the calf killed on day 23 were moderately
enlarged and slightly edematous. The hemal lymph nodes were nor
mal in size. The thymus was small, but of normal consistency.
One small abcess containing thick greenish exudate was present in
1 tonsil. Red bone marrow extended half way down the femoral
shaft, contrasting with red marrow that occupied approximately the
proximal 20% of-the femur in calves killed previously.
The lymph nodes of the calf killed on day 30 were slightly
enlarged and edematous. One prescapular lymph node contained
The hemal lymph nodes were greatly enlarged.multiple ecchymoses.
The thymus was not evident. The abdominal cavity contained ap
proximately 40 cc. of amber fluid.
An experimental calf in a weakened condition died on day 37
after routine blood samples were taken. Although not detected on
external examination of this calf, the connective tissue of the
All of the lymphintcrmandibular space was found to be edematous.
nodes were moderately enlarged and edematous and hemorrhages were
present in the prescapular and renal lymph nodes. The thymus was
small and edematous. The epicardial surface contained multiple
petechiae, the right ventricle was rounded and there was serous
atrophy of pericardial fat. The pericardial sac contained ap
proximately 30 cc. of amber fluid. The liver capsule and parench
yma were slightly more difficult to cut than normal. The kidneys
had multiple petechine on'their surfaces and there was serous
atrophy of-perirenal fat, The cortices of the adrenal glands
25
were congested.
The calf killed on day 38 was weak, and its eyes were sunken.
Submandibular edema was conspicuous. All of the lymph nodes were
moderately enlarged and edematous, and 1 prefemoral lymph node
had several ecchymotic hemorrhages. The hemal lymph nodes were
greatly enlarged and prominent, and the thymus was small and
edematous. Depot fat in the animal was minimal, The cut surface
of the ventricular myocardium was pale, and the pericardial sac
contained approximately 10 cc. of amber fluid. The liver was pale,
and the capsule and parenchyma were more resistant to cutting
than normal. The abdominal cavity contained approximately 25
cc. of amber fluid. The capsular surfaces of both kidneys con
tained multiple petechiae. The thymus was gelatinous in consis
tency.
The lymph nodes of the calf killed on day 45 were moderate
ly enlarged and edematous, and the hemal lymph nodes were greatly
enlarged. Although no free fluid was observed in the body cavi
ties, considerable fibrin was present in the ventral abdomen. The
mesentery of the spiral colon contained a large quantity of edema.
Serous atrophy of fat was evident. The thymus was small and
edematous. The right ventricle of the heart was moderately
rounded. The apical lobes, the ventral portions of the cardiac
lobes and small ventral portions of the diaphragmatic lobes of
the lung were red and firm. The trachea contained white foam.
The hepatic capsule and parenchyma were moderately resistant to
26
cutting. Red bone marrow extended more than half way down the
length of the femur.
The last calf was killed on day 88. This calf had chronic
cystitis. The gross, microscopic and clinical pathological data
did not reflect uncomplicated chronic trypanosomiasis, therefore
data from this calf are not included.
Microscopic Pathology
Lungs
Within 12 hours after inoculation, hypertrophy was evident
in the endothelium of the pulmonary arterioles. There appeared
to be an excess number of mitotic figures in the endothelium of
arterioles. Within 24 hours post inoculation some alveolar spaces
,contained serofibrinous exudates and erythrocytes. Intraaiveolar
aggregations of macrophages were seen by day 8, and in sections
of lung tissue collected from calves killed subsequently, there
were similar aggregations of macrophages in blood vessels. Some
small blood vessels observed in longitudinal section contained
multiple small dark bodies. By day 23 some alveolar walls were
slightly thickened and contained eosinophilic material. The
interalveolar lesion became more prominent with time (Fig. 5).
This lesion was randomly distributed throughout the parenchyma
within pulmonary lobules. Typically, an affected lobule was
located adjacent. o a normal lobule, and therefore the affected
tissue was dispersed in a random pattern. By the end of the
27
Fig. 5. Lung from calf infected 38 days previously with Innumerable debris-laden macrophages ac-Trypanosoma vivax.
counted for most of the increased thickness of the interalveolar
tissue. No deposition of extravascular material was demonstrable
with special stains. H&E. 350X.
28
experiment the interalveolar tissue was seen to contain numerous
macrophages, some of which apparently contained engulfed material.
Lung sections collected on days 30, 37 and 38 and processed with
Periodic Acid Schiff or Masson's Trichrome Stain* indicated the
basement membranes of the vessels were normal, and there was no
evidence of deposition of excess material along the vessels.
Heart
Evidence of progressive changes were found in the myocardium
as early as day 2. At that time small lymphocytes had infiltrated
the interstitial tissue around blood vessels and lymphatics.
Larger lymphocytes were recognizable by day 5 and day 12. The
endothelial cells of arterioles were hyperplastic. After a month
there were many plasma cells in the interstitial tissue along the
blood vessels, lymphatics, nerves and between muscle bundles
(Fig. 6). Some myocardial nuclei were altered, with the chromatin
being arranged in a long, thin, wavy line along the central axis
of the cell, such as is described for the "caterpillar cell" of
certain human cardiac myopathies (43). Sarcosporidia were common
in heart muscle as well as in skeletal muscle.
Liver
Initial changes in the liver were observed by day 3. There
*PAS & Hasson'sTrichrome Stains: AFIP Protocol. 1957
29
Fig. 6. Heart from calf infected 38 days previously with Trypanosoma vivax. Cellular infiltration at this stage invclved
numerous plasma cells as well as lymphocytes. Some myocardial nuclei resembled "caterpillar nuclei" seen in certain human myopathies. H&E. 150X.
Fig. 7. Liver from calf infected 38 days previously with Trypanosoma vivax. Periacinar congestion masked an occasional
dying hepatocyte, Fatty metamorphosis was predominant in midzonal areas. H&E. 130X.
30
were areas of acute focal necrosis which were infiltrated with
neutrophils. Some of the hepatocytes located near terminal he
patic veins had undergone fatty metamorphosis. A few lymphocytes
had infiltrated into portal tracts by day 5, and they were more
numerous by day 8. By day 23 there were large aggregations of
macrophages in terminal hepatic veins, and a few neutrophils had
infiltrated into the parenchyma. Kupffer cells scattered through
out the tissue were filled with globules of hemosiderin. Peri
acinar congestion was marked by the fifth week, and in most, but
not all calves there were many degenerated hepatocytes around
terminal hepatic veins, with fatty metamorphosis being present
in the midzonal regions (Fig. 7). Periacinar fibrosis could not
be demonstrated by special staining techniques.
Kidneys
In the kidneys of the calf killed on day 3 there was exces
sive proteinaceous material in the proximal convoluted tubules,
and the glomerular tufts were slightly hypercellular. The degree
of proteinuria had decreased to a low but persistent level by the
end of the second week. Endothelial hypertrophy was evident in
arterioles oriented nearglomeruli. The lumens of some of these
arterioles were very small during the second week. Perivascular
infiltration of lymphocytes, plasma cells and other mononuclear
cells becaeeuore prominent as time elapsed. At the end of 3
weeks, large accumulations of cells were present around the
31
arcuate arteries.
The degree of hypercellularity of the glomerular tufts in
creased and at the end of 5 weeks the tufts completely filled
some of the Bowman's spaces. Macrophages filled with hemosiderin
were located predominently in the medulla. Accumulations of
neutrophils in the proximal convoluted tubules and elsewhere in
the kidney were visible on day 38.
Lymph nodes
In general, by 12 hours post inoculation mitotic activity was
at a high level in the germinal centers of most lymph nodes. Dur
ing the experiment most lymph nodes had evidence of lymphoid or
reticuloendothelial hyperplasia, however, toward the end of the
experiment some germinal centers appeared exhausted. Occasionally
small hemorrhages and edema were seen. Macrophages containing
hemosiderin were scattered throughout the lymphatic tissue during
the latter portion of the experiment.
The mediastinal lymph nodes were typical of those nodes in
which lymphoid hyperplasia occurred. During the first week there
was a moderate degree of hyperplasia and many necrotic cells were
present in germinal centers. In the second week there apparently
was an increase in the number and size of the germinal centers.
Harked lymphoid hyperplasia was evident during the last 4 weeks
of the experiment.
In the prescapular lymph nodes a mild reticuloendothelial
32
cell hyperplasia was evident by the end of the first week, and the
condition developed to a moderate or marked level during the sec
ond week. The condition regressed considerably near the end of
the experiment. Concurrently, the concentration of lymphocytes
appeared slightly decreased oh day 2 and on day 12 there was
moderate depletion, although the concentration uf lymphocytes was
approximately normal at the end of the experiment.
Bone Marrow (femur)
Specimens of bone marrow from the first 2 calves killed con
sisted mostly of depot fat with some evenly distributed hemo-
After day 3 the blood forming tissue increased atpoietic cells.
the expense of the fatty tissue and by day 12 there were confluent
areas of hemopoietic tissue. Cells of the erythroid series were
It wasgenerally more numerous than cells of the myeloid series.
estimated that there were about twice as many erythroid cells as
myeloid cells on day 23, After this time reticuloendothelial
(RE) cells were numerous, and in some areas they had aggregated
in discrete foci. Congestion was apparent in tissue collected
after 3 weeks, and tissue collected on day 38 contained many
cells of the neutrophilic series.
Ear
Endothelial hyperplasia was evident in a few subcutaneous.
arterioles by day 3, but this was not evident in calves killed
33
toward the end of the experiment. A slight to moderate infiltra
tion of eosinophils, lymphocytes and macrophages had occurred in
the interstitial tissue along blood vessels and lymphatics. The
concentration of infiltrating cells in the subcutaneous tissue
remained at a fairly constant level during the course of the dis
ease,
Thymus
During the first week there was a slight increase in numbers
of eosinophils and neutrophils in the interstitial tissue between
lobules. The cortices of the lobules appeared thin by day 12,
and by day 23 there was a definite depletion of thymocytes. No
parenchyma remained by day 30, and on days 37 and 38 all that
remained was supporting connective tissue and vessels, with no
distinctly discernable boundary between cortex and medulla.
There was a moderate degree of atrophy present in some of the
thymus tissue collected on day 45 but in other areas the tissue
was normal.
Spleen
There was moderate congestion and moderate to marked hemo
siderosis in sections of spleen collected after the first week,
and this condition persisted throughout the experiment. Neutro
phils collected in the red pulp, mostly near Malphigian corpus
cles, in the late stages of the disease.
34
Hemal Lyiaph Nodes
There was a variable degree of congestion.
Fluorescent antibody stained tissue
Trypanosomes fluoresced with a typical apple green color in
smears from infected blood (Fig. 8). Smears were improved by re
moving most, but not all, of the erythrocytes as less time Oas
required per slide. The optimum dilution of the conjugate was
1:50.
Trypanosomes with somewhat less fluorescence than that of
the controls were detected in the mediastinal lymph node and
spleen of the experimental calf killed on day 23. The parasite
seen in the mediastinal lymph node was intact, and the nucleus
and kinetoplast both had\a strong fluorescence. The rest of the
parasite was easily visible, but fluorescence was weak. The sec
cond trypanosome, seen in splenic tissue, possessed similar
fluorescing qualitiesas the one described in the lymph node.
It was intact except for a small portion of the anterior ex
tremity. In neither case was it possible to say if the parasites
were located in vessels. Because the organisms were essentially
of the same shape as ,the trypanosomes observed in blood films,
they probably were lying free in the interstitial fluid. Almost
certainly they were not intracellular. An examination of tissue
cut from the samq.,frozen blocks and examined by'light microscopy
35
Fig. 8. Blood smear from Trypanosoma vivax infected calf incubated 30 minutes (37 C) with FITC-conjugated immune serum. Large oval nucleus and smaller kinetoplast (left extremity of organism) exhibited identical quality of apple green fluorescence. Three erythrocytes are present. Ultraviolet light with dark field condenser. 1620X.
36
failed to locate any portions of parasites.
Microscopic structures having an evenly distributed apple
green fluoresctince were observed in tissue from certain organs of
the experimentally infected calves but not from tissue of the con
trol calf. In cardiac tissue collected at 12 hours a blood vessel
was seen to be surrounded by an area of evenly distributed flu-
In renal tissuG taken later in the experiment thereorescence.
was a similar type of fluorescence that was evenly distributed
through the glomerular tufts of some Bowman's spaces and in the
interstitial tissue around vessels associated with the convoluted
tubules and to a lesser extent with descending tubules.
In tissue from the control calf and most infected calves
non-specific fluorescence was seen in the form of multiple, even
sized circular granules which were not trypanosomes. The granules
had an apple green fluorescence and were sufficiently small that
as many as 200 were estimated inside some cells. The cells were
suspected of being macrophages. Usually, however, the number in
a cell was much less, although the size of the granules remained
fairly constant. Organs most often affected included lymph nodes,
spleen and thymus, while the cerebrum was rarely involved.
Granules similar to those seen intracellularly were observed
intercellularly in bone marrow of at least 1 infected calf. In
some areas the granules were packed tightly together in the inter
cellular spaces and often were sufficiehtly numerous as to form a
syncytium which tended to outline the normal cellular structures.
37
CHAPTER V
DISCUSSION
Calves inoculated with I. vivax developed fever within 24
hours, which was similar to results reported with 1. vivax (15,
30) and T. conaolense (13) in Africa, while inoculation of T.
vivax in Venezuela rarely caused fever (34). During the rest of
the experiment the average morning and afternoon rectal tempera
tures did not deviate markedly from the normal range, which also
was similar to the African work. It was difficult to demonstrate
relapsing temperatures with T. congolense infections except by
calculating a 3-day average in order to "smooth" the temperature
curve, or else by using a virulent strain of parasite in cattle
of very young age (13, 15). It is well known that trypanosomes
undergo changes in antigenic composition (27), which may be re
lated to the decrease in fever. It is possible that a mild per
sistent fever did occur which was sufficiently variable as to be
indistinguishable from normal individual variation of rectal tem
perature in a small group of animals.
The afternoon temperatures were consistently higher than
those recorded in the morning, with the exception of the initial
fever, because the calves were regularly exposed to strong after
noon sunlight. Some rectal temperature values were low near the
end of the experiment because I or more calves were in a moribund
state.
38
Each calf reacted similarly, gradually becoming weak and
listless, although its appetite remained normal. Submandibular
edema, which developed during the latter part of the experiment,
probably was related to the failing heart and to decreased os
motic pressure of the blood due to loss of albumin and possibly
other blood proteins via..the kidney. Albumin is c6nsidered to
account for 45. or more of the osmotic pressure of the blood and
is important in fluid balance (9). The edema tended to regress
as death became imminent, and its appearance, followed by re
gression was interpreted as an unfavorable prognostic sign. It
was likely that dehydration in the terminal stage was a factor in
the regression of the edema, although quantitation of water intake
was not monitored. A similar report of submandibular edema was
made in the 1. vivax infected cattle in Venezuela (34), but not
in the African work (15, 30).
Anemia developed rapidly in the acute phase, based on the
rapid rate of decrease of hemoglobin and PCV (Fig. 1). In the
subacute phase these blood parameters decreased at a persistent
but slower rate which appeared to be directly associated with
the gradual development of emaciation. Anemia, rather than di
lution due to an increase of plasma volume, was suggested by
concurrent evidence of an erythropoietic response in histologi
cal sections of femur marrow and smears of rib biopsies and a
slight but immediate increase in total and direct bilirubin
levels following inoculation (Fig. 3).
39
When the PCV reached the range of 16% to 20%, death could be
expected, as illustrated by a PCV of 17% in the calf that died.
Rave, in Colombia (39), infected 5 calves of similar age and
breeding to the calves used in this experiment and observed anemia
in which the PCV decreased to 15% to 22%, after which a calf had
to be killed on day 47. Except in cases where animals died in
the acute phase, anemia was one of the most constant signs that
developed in cattle infected with I. vivax (15, 30, 34) and
anemia, rather than hypoglycemia, was suspected of being the
cause of death in subacute bovine trypanosomiasis in Kenya (30).
Anemia of Venezualian cattle infected with I. vivax was often
aggrevated by blood loss from chronic intestinal parasitism (34).
The present experiment indicated that trypanosomiasis in Colom
bian calves under favorable husbandry conditions and in the ab
sence of other hemoparasites can be fatal, whereas apparently
other native calves infected with T. vivax are often clinically
unaffected (50). It is evident that innate resistance, husbandry
practices and perhaps unknown factors are also important.
The timing of increases in serum bilirubin levels coincided
with that of the first parasitemia, and apparently was the re
sult of the release of hemoglobin from erythrocytes. However,
the bilirubin levels did not exceed the limits of published
normal values for calves (7). The increase in direct or con
jugated serum bilirubin was indicative of normal hepatic function
in response to the elevated level of non-conjugated bilirubin.
40
The latter is the difference between the total and the direct
bilirubin, and this had-increased approximately 2 fold with the
first parasitemia. In contrast, data derived from African calves
infected with 3. congolense and which were anemic had normal
icterus index values, although no serum bilirubin tests were
made (13).
The SGOT values increased gradually from day 23 until the
experiment terminated (Fig. 3), and histologic examination re
vealed that lesions in cardiac and hepatic tissue had developed
It is known that homogenates of T. vivax reduring this time.
lease 10 times as much SOOT as is present in equal volumes of
normal serum and that the SOOT is elevated in serum from animals
that have experienced destruction of muscle and hepatic tissue
The rise of SOOT in the chronic phase probably was due(26).
chiefly to tissue necrosis. It is possible that the slight but
rapid rise in enzyme levels during the 24 hours following inocu
lation was due to enzymes released from the infecting trypano
somes. The similar rate and magnitude of SGOT increase after
day 5 that coincided with the first parasitemia, and that was
followed by a slightly elevated plateau, was probably due at
least in part to the periodic destruction of trypanosomes. The
tissue desruction that occurred in the chronic phase evidently
released sufficient tissue enzymes to raise the level higher,
!although the absolute rise was indicative of only mild tissue
damage. Additional evidence of hepatic tissue damage was furnish
41
ed by the slightly elevated levels of serum sorbitol dehydro
genase (SDH), an enzyme specific for hepatic tissue (19), during
the third and fourth weeks (Fig. 3).
There apparently was considerable damage to the endothelium
of the renal glomeruli due to trypanosomes of the first parasitem
ia and succeeding parasitemias, because there was evidence of a
moderate degree of proteinuria in histological sections of kidney
as well as a decrease in total serum proteins (Fig. 3). Total
serum proteins stabilized at a lower level during the second week
which likely was the result of several factors, including a re
duced rate of loss of proteins through the kidney. The decreased
level of proteinuria observed in histologic sections of renal
tissue after the second week supported this theory. The extent
of hepatic lesions was considered insufficient to account for the
decrease in production of serum albumin.
There was a slight decrease in total serum globulins, but
the percentage of total serum globulins was greater because of the
loss of serum albumin. It is possible that some of the smaller
alpha globulins also were lost via the kidney, as there was a
very slight decrease of this fraction. The first proteins to
leak through the glomerular membrane are albumin and alpha glo
bulins, and if the damage is severe, the beta and gamma fractions
also may be lost (3). The A:G ratio decreased slightly in the
first 2 weeks and this change was apparently due to the consider
able loss of albumin through the kidney rather than due to an
42
absolute increase of globulins.
These results agree with results from . vivax infections
in sheep (6, 40) and L. brucei in rats (31) in which there was
a decrease in serum albumin. One author (31) attributed the
loss to kidney damage, while another (6) stated that a primary
increase of serum globultn acted as a plasma expander, resulting
in dilution of albumin and beta globulins. Total serum proteins
decreased and the A:G ratio w4s reversed, with excess albumin
appearing in the urine of rabbits with T. evansi (47).
Gamma globulins increased gradually in the 4 weeks following
the first parasitemia (Fig. 3), and this was probably related to
the immune response following the protozoal infection. Similar
responses were observed by others in trypanosomiasis (6, 31, 40).
The increase of gamma globulins is accepted as being related to
the development of immunity following exposure to infectious
agents, although the magnitude of response to protozoal agents
may not be as clear cut as that of bacterial infections (9).
Hoiever, in calves with eperythrozoonosis, another hemoprotozoan
disease, the serum gamma globulins actually decrease and the
A:G ratio increases, which is contrary to most reports (10).
The same individual calf had progressively lower blood glu
cose values on each of the last 2 observation days (Fig. 4), and
at necropsy there was emaciation and serous atrophy of fat.
Liver tissue showed moderately increased resistance to cutting
and microscopically there was hepatic periacinar congestion but
43
no fatty metamorphosis. Fiennes (13) found no si~nificant
changes in levels of blood sugar in calves or adult cattle with
1. conaolense infections, although he indicated that hypoglycemia
had been reported occasionally in cattle with chronic trypanoso
miasis.
A recent report suggested that T. brucei infection in rats
resulted in a diabetes mellitus-like condition (48). In the ratb
there was a decrease of peripheral glucose utilization in the
acute stage that was masked by a high rate of glucose consumption
by the trypanosomes, however, hypoglycemia became apparent in the
.chronic stage. The condition responded to, but was not entirely
restored to normal by insulin. The response described is more
characteristic of adrenal diabetes than of pancreatic or pituitary
diabetes (28). In this experiment, only 1 calf developed hypo
glycemia, and no evidence of fatty metamorphosis was observed in
the liver of this particular calf, a lesion consistently reported
with diabetus mellitus (33).
Leucocytic parameters in trypanosomiasis apparently vary
with the age of the cattle, species of the trypanosome, suscepti
bility to other diseases and unknown factors. In this experiment
neutropenia occurred following the first parasitemia, but neutro
philia developed after 3 weeks (Fig. 4). This data correlated
with the presence of pus in the convoluted tubules of the calf
killed on day 38 and with chronic cystitis seen in the last ex
perimental calf which was killed on day 88. Apparently an in
44
ctease in susceptibility to other diseases developed before the
end of the experiment, probably because of a general decrease
in body defenses. Mild neutropenia was also reported in adult
African cattle infected with either T. vivax (16) or 2. congolense
(13), whereas Colombian calves infected with T. vivax developed
mild neutrophilia (39). -
There was mild lymphocytopenia after the first week (Fig. 4),
although histologic examination of most lymph nodes revealed an
increased level of activity in germinal centers and increased
numbers of lymphocytes in the sinuses. Histological examination
revealed an infiltration of lymphocytes into the interstitial
tissue along blood and lymphatic vessels in most organs, appar
ently in response to antigens, possibly including soluble antigens
released from the trypanosomes. In contrast, lymphocytosis was
reported in calves infected with 1. congolense (13) or T. vivax
(39), although the number of calves used in the latter work was
small.
There was generalized hypertrophy of endothelium and sten
osis in the small blood vessels. The many small dark bodies
observed in small blood vessels of the lung and otheq organs
were suspected of being degenerated trypanosomes. Lymphocytes,
plasma cells and macrophages were common in interstitial tissue
along blood and lymph vessels.
Material suspected of being dead trypanosomes was seen in
stained sections of the lung. Since the lung contained the first
45
capillary bed in the path of the trypanosomes following inocu
lation in the jugular vein, a portion of the initial inoculum
probably became lodged in pulmonary tissue. Alterations in the
integrity of the vascular endothelium and hemorrhage apparently
resulted from the influence of the parasites. Whatever the
process there was a gradual thickening of the interalveolar struc
tures which was easily detectable after 3 weeks (Fig. 5). This
change was not consistent throughout the parenchyma but developed
in individual scattered areas, probably as a function of the
distributibn pattern of dying trypanosomes in the pulmonary
parenchymna. It is possible that the trypanosomes of succeeding
parasitemias became lodged in vessels already damaged or sensi
tized and provided a continuous source of irritation of tissue
in focal aroas. Another possible source of continuing irritation
may have been from soluble antigens, which are exoantigens, and
are known to develop in certain T. vivax infections (25).
Apparently the interalveolar change was due principally to the
numerous macrophages observed, many of which contained engulfed
material. Based on the examination of lung tissue using apecial
staining techniques,* there was no evidence of the deposition of
excess amorphous material associated with interalveolar capillar
ies.
The efficacy of gaseous exchange between the alveoli and the
*PAS and Massonlo Trichrome Stains: AFIP Protocol, 1957
46
surrounding capillary network probably was decreased due to ac
cumulations of macrophages along interalveolar capillaries. Other
conditions contributing to anoxia were the anemia and cardiac in
sufficiency. The combined influence of these 3 factors probably
deterimentally influenced endothelial and myocardial viability.
The hearts from some of the calves killed last had enlarged
This probably was due chiefly to pulmonaryright ventricles.
hypertension caused by atenosis of pulmonary arterioles and also
by the accumulation of macrophages along interalveolar blood
Apparently the parasites exerted a deterimental effectvessels.
on the endothelial component of myocardial vessels in a manner
similar to that observed in the lung, consequently, inflammatory
cells were attracted into the interstitial tissue surrounding
blood vessels and lymphatics (Fig. 6). This inflammatory process
was more pronounced in the myocardium than in other organs.
Plasma cells were prominent in cardiac and lymphatic tissue after
Plasma cells in large numbers are usually limited to1 month.
the lymph nodes, and they are particularly associated with chronic
L '
mental inoculation of T. vivax into sheep IgG immunoglobulins
appeared, whereas prior to that time the immunoVnetic response
inflammation (18). It is interesting that 1 month after experi
had consisted of only macroglobulins (40). Changes observed in
the myocardial nuolei of chronically affected calves resembled
the Anitschkow myocytes or "caterpillar cells" tihich have been
described for human cardiac myopathies (43). Cardiac insuffi
47
ciency probably accounted for the chronic passive congestion of
the liver and contributed to the submandibular edema and edema
of the lymph nodes and other organs. However, the hypoproteinemia
probably contributed to the edema also due to decreased osmotic
pressure of the blood.
It is possible that.-the acute focal hepatic necrosis observed
on day 3 was due to trypanosomes becoming lodged in the sinusoids.
However, similar lesions were not present in the liver of calves
killed later, although parasitemias were repeatedly demonstrated.
The periacinar congestion was probably a result of impaired func
tion of the heart. Zones of degenerating hepatocytes observed
around the terminal hepatic veins and fatty metamorphosis seen in
midzonal hepatocytes (Fig. 7) were also probably due to abnormal
circulation, although the anemia and possibly the deposition of
material around pulmonary capillaries may have been contributing
factors. Mild to moderate lymphocytic infiltration of portal
areas was similar to the general lymphocytic infiltration of
interstitial tissue in other organs. Aggregations of macro
phages were seen in terminal hepatic veins. Fiennes similarly
described periacinar congestion, fatty metamorphosis and aggrega
tions of macrophages in hepatic vessels in African cattle in
fected with 3. congolense (14) and another investigator found
large macrophages in the ear capillaries and venules of rabbits
2 weeks after being infected with ,,brucei (23).
,Tieintegrityof, the vessel walls of the glomerular tdf ii
48
was altered before the first parasitemia became evident and pro
teinuria'developed. This change was apparently initiated by the
relatively small number of inoculated trypanosomes. Hyperplasia
of the cells of glomerular tufts eventually resulted in filling
Bowman's spaces. Some of the tufts were embedded in a matrix of
proteinaceous material. .Neutrophils were observed in the renal
tubules from the animal killed on-day 38 and the last calf re
maining alive, which was killed on day 88, had a chronic purulent
cystitis. It is tempting to explain the decrease in serum al
bumin by the loss of protein through the kidneys. Macrophages,
lymphocytes and plasma cells collected around arterioles and
small renal arteries. Some of these perivascular infiltrations
were of considerable size at the end of the experiment. Cortical
and arcuate vessels were affected most frequently.
The hyperplasia of RE and lymphatic elements of lymph nodes
was assumed 'toresult chiefly from the trypanosomal infection.
The plasmacytosis which developed in certain lymph nodes during
the fifth week coincided with the appearance of high numbers of
plasma cells in the interstitial tissue of the heart. However,
the reason for the plasmacytosis at that specific time was not
determined.
Hypertrophy of femoral bone marrow was evident at necropsy,
and histologic examination of marrow indicated that a response
in erythropoietic activity had begun'during the,first week post
inoculation. ,This response coincided with the time of the first
49
parasitemia and with the initial decline in hemoglobin after
day 5. Increased erythropoietic activity continued thereafter
and there was no evidence of exhaustion of the hemopoietic sys
tem. The M:E ratio (Fig. I),based on rib biopsies, declined
moderately during the experiment, and this change was considered
due principally to increased erythropoietic activity. The pro
liferation of RE tissue in bone marrow was considered evidence
of an extreme reaction by the body defense mechanism in response
to a persisting infection.
There'are few reports in the literature describing bone
marrow response in trypanosomiasis. Fiennes found that the marrow
throughout the full length of long bones of calves and adults in
fected with 1. congolense was red, but stated that in histologi
cal sections the marrow was aplastic. This redness probably
indicates a state of congestion of a tissue which normally is
filled largely with depot fat and hematopoietic tissue.
No immediate response was observed in the granulocytic cell
series in bone marrow sections in response to decreased levels
of circulating neutrophils. However, there was evidence of
stimulation of the neutrophilic cell series in bone marrow sec
tions from the last 3 calves killed which correlated with the
increased numbers of circulating neutrophils in the same calves
(Fig. 4).
In the ear endothelial hyperplasia of some subepithelial
arterioles had occurred by day 3 but stenosis was not observed.
50
moderate degree of cellular infiltration of inter-There was a
stitial tissue around blood vessels but it did'not reach the
intensity of the perivascular infiltration that occurred in the
A portion of the infiltrated cells werekidney and heart.
eosinophils, whereas in the other organs except the thymus the
The influence of factorsnumber of eosinophils was minimal.
other than the trypanosome infection could not be ruled out.
Accumulations of neutrophils were observed in the red pulp
of the spleen in the last several calves killed. The effect was
most pronounced in tissue near the splenic corpuscles. The
neutrophils possibly were attracted to that site as a result
of antigens which accumulated in cells of the RE system, al
though normal pooling of cells due to decreasing circulation can
not be ruled out.
Fluorescent antibody conjugate at a dilution of 1:50 was
More dilute preparations resulted in inconconsidered optimum.
sistent fluorescence of the trypanosomes and less dilute prepara
tions resulted in excess non-specific fluorescence.
Positive control blood smears containing a reduced number
of erythrocytes were preferred because less time was required
in locating the trypanosomes, whereas the presence of a few
erythrocytes facilitated initial focusing of each slide.
The color and intensity of fluorescence of the nucleus and
kinetoplast within a given trypanosome was identical (Fig. 8)
suggesting that components common to both structures were
51
involved. This effect applied both to the trypanosomes sean in
positive control blood smears and in trypanosomes from infected
tissue, although the fluorescence was less intense in the latter.
Deoxyribonucleic acid (DNA) and nucleoproteins of DNA are known
to be present in both the nucleus and kinetoplast of trypanosomes
(11) and both compounds are of sufficient size as to stimulate an
immune response. It is likely that one or both compounds served
as antigens common to both the nucleus and kinetoplast.
Fluorescence of the external surface of the trypanosomes was
of a much more subdued intensity compared to the fluorescence of
the nucleus and kinetoplast. The external antigens involved were
probably either a part of the external wall or were related to
metabolic products that may have coated the organisms. Apparent
ly the external antigens were less recognized as foreign antigens
by the immune mechanism of the calves as compared to the endo
antigens from the nucleus and kinetoplast. Metabolic excretory
products of trypanosomes are likely to be of small average molec
ular size, and therefore the antigenic response to such products
would be relatively weak.
The amorphous, non-particulate fluorescence observed in glo
merular tufts, convoluted and descending tubules and intersti
tial tissue in the kidney was not seen in renal tissue of the
control calf, It is possible that the fluorescence war ralated
to soluble antigens to T. vivax which have been demonstrated
(25,,49). Such antigens would likely be widespread in the,
52
tissues and this may explain the diffuse distribution of fluor
escence that was observed. The pattern of distribution of
fluorescence in interstitial tissue along vessels was thought
to be similar to the distribution of inflammatory cells seen
with conventional microscopy. The source of the antigens was
suspected of being the degenerated trypanosomes in or near small
blood vessels or lymphatics.
Fluorescence of a particulate, granular nature was considered
to be non-specific because a similar pattern of fluorescence was
seen in tissue of the control calf. The number of fluorescing
particles was highest in organs containing an abundance of macro
phages, such as the spleen and lymph nodes, and therefore the
fluorescence may have been related to breakdown products. How
ever, granples believed to be of a similar nature because of
their shape, size, and degree of fluorescence were also abundant
in intercellular areas of the bone marrow.
53
CHAPTER VI
SUMMARY
T. vivax obtained from a clinically sick cow near Neiva,
Colombia, South America was passed in a sheep and in a calf, and
then inoculated into the.jugular vein of 14 Holstein-Friesian
calves. Fever occurred by 24 hours and recurring parasitemia
commenced after 72 hours post inoculation. It was estimated
that practically all of the 14 calves would have died spontan
eously of trypanosomiasis within 3 months if none of the calves
had been euthanitized.
Associated with the first and subsequent parasitemias were
decreases in hemoglobin, PCV, M:E ratio, serum albumin, A:G ratio
and neutropenia, and slight increases in total and direct bili
rubin and SGOT levels. Later changes included slightly elevated,
fluctuating total bilirubin levels, slightly increased SGOT and
gamma globulin levels and slightly decreased alpha and beta
globulin levels. Blood glucose levels decreased in only 1 calf
after day 30.
After 2 weeks all calves exhibited a gradual loss of body
condition and later submandibular edema usually became evident.
The latter condition sometimes regressed and this sign along with
a PCV below 20% indicated that death was near. After 4 weeks the
post mortem lesions'considered sufficiently consistent to be of
diagnostic'value included conspicuously hypertrophied, edematous
54,
lymph nodes and hypertrophied hemal lymph~nodes, emaciation,
rounded right heart, palpably firm liver, atrophied thymus and
hypertrophied femoral bone marrow.
Associated with the 1. vivax of the infecting inoculum and
of succeeding parasitemias was a generalized endothelial hyper
trophy and mononuclear cell infiltration along blood and lymph
vessels, with proteinuria and bone marrow hyperplasia. General
ized lymphatic and RE hyperplasia occurred, but was not proven
At 3 weeks post inoculationto be due to the trypanosomiasis.
there was an aggregation of macrophages containing engulfed ma
terial distributed along capillaries in pulmonary interalveolar
tissue, and this lesion in combination with the anemia and ap
parent cardiac insufficiency were thought to be important in
the development of anoxia and probably contributed to the sin
gle fatality observed.
Periacinar congestion and fatty metamorphosis of midzonal
hepatocytes were probably related to the failing heart and in
directly to the pulmonary lesion. Cystitis and pyelonephritis
in the last 2 calves to be killed probably was a reflection of
generalized debility due to chronicity of the trypanosome in
fection.
The detection of intact trypanosomes or their residue in
histologic sections, utilizing a direct fluorescent antibody
Generalized fluorescence oftechnique, was ,of limited success.
interstitial tissue associated, at least in part, with vessels
55
was observed in tissue from infected calves but not from the
control calf. It was suspected that the fluorescence was re
lated to the presence of soluble antigens to the trypanosomes,
56
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61
VITA'
Charles Arthur'Daley, son of Thomas Henry Daley and Lena
Gunderson, was born near Shelby, Montana, on March 25, 1932.
He attended elementary and high school in Shelby, Montana, and
received his diploma in May, 1951.
He entered Montana State University in Sept., 1951 and
graduated in June, 1956 with a B. S. degree in range management
and a M. S. degree in/animal industry. At that time he entered
the U. S. Air Force, and was released in Jan., 1960.
In Sept., 1960 he entered Washington State University and
was graduated inMay, 1964 with the D. V. M. degree. He then
was employed by tho Montana Livestock Sanitary Board until Aug.,
1968, working in regulatory and diagnostic veterinary medicine,
In Sept., 1968 he accepted a fellowship from the Institute
of Tropical Veterinary Medicine, Texas AM University, with
financial support from U.S.A.I.D. and The Rockefeller Foundation
to work toward the M. S. degree in veterinary pathology at Texas
MM University and Bogota, Colombia, South America.
He was married on Apr. 5, 1968 to Dorothy Ann Schanil of
Bozeman, Montana.
The typist for this thesis was Charles Arthur Daley,
College StationTexas.